感染病學(xué)基礎(chǔ)-病原學(xué)診斷-8-30

病原學(xué)診斷

Diagnosticmicrobiology內(nèi)容Contents

1.早期開展簡(jiǎn)史2.顯微鏡術(shù)(Microscopy)3.體外培養(yǎng)(Invitroculture)4.分子診斷(moleculardiagnosis)5.血清學(xué)診斷(serologicdiagnosis)6.動(dòng)物實(shí)驗(yàn)(animalexperiment)1674，AntonyVanLeeuwenhoek借助自制透鏡發(fā)現(xiàn)微小生物體1859,LouisPasteur制備液體培養(yǎng)基1881,Koch和Hesse制備固體培養(yǎng)基1949,JohnFranklinEnders建立動(dòng)物細(xì)胞培養(yǎng)技術(shù)……1.早期開展簡(jiǎn)史〔1〕目的：直接檢測(cè)微生物〔細(xì)菌、真菌、寄生蟲蟲卵幼蟲和成蟲、感染的細(xì)胞中的病毒包涵體〕;初步或明確鑒定微生物2.顯微鏡術(shù)(Microscopy)初步鑒定：多數(shù)細(xì)菌明確鑒定：真菌和寄生蟲；促進(jìn)明確鑒定：熒光抗體或其它標(biāo)記物染色后，顯微鏡觀察〔2〕技術(shù)：普通光學(xué)顯微鏡術(shù)(Brightfieldorlightmicroscopy)暗視野顯微鏡術(shù)(Darkfieldmicroscopy)熒光顯微鏡術(shù)(Fluorescentmicroscopy)相差顯微鏡術(shù)(Phase-contrastmicroscopy)電子顯微鏡術(shù)〔Electronmicroscopy)普通光學(xué)顯微鏡術(shù)(Brightfieldorlightmicroscopy)：細(xì)菌染色標(biāo)本活菌運(yùn)動(dòng)大的微生物如寄生蟲和真菌病毒感染的病變細(xì)胞和包涵體組織病理暗視野顯微鏡術(shù)(Darkfieldmicroscopy)：不染色活菌螺旋體及其動(dòng)力熒光顯微鏡術(shù)(Fluorescentmicroscopy)：各種微生物用熒光素標(biāo)記后發(fā)出熒光相差顯微鏡術(shù)(Phase-contrastmicroscopy)：可觀察到微生物的內(nèi)部結(jié)構(gòu)電子顯微鏡術(shù)〔Electronmicroscopy)：常用做研究工具直接觀察到高濃度液體中的病毒顆粒的形態(tài)和大小、低濃度液體可離心后收集沉淀觀察或免疫電鏡觀察微生物的超微結(jié)構(gòu)〔3〕檢查方法：直接檢查〔directexamination)鑒別染色〔differentialstains，eg.Gramstain)抗酸染色〔acid-faststains)熒光染色〔fluorescentstains)〔1〕成功取決于：微生物的生物學(xué)特性感染的部位患者對(duì)感染的免疫反響適宜培養(yǎng)基〔三種類型〕和條件3.體外培養(yǎng)(Invitroculture)培養(yǎng)基類型〔細(xì)菌和真菌〕培養(yǎng)基類型〔衣原體、立克次體和全部病毒〕：細(xì)胞培養(yǎng)〔1〕分子種類：DNARNA蛋白質(zhì)〔2〕優(yōu)勢(shì)：適宜于不能別離或免疫學(xué)檢查的微生物檢查；檢測(cè)基因型或突變〔eg.耐藥〕敏感、特異和平安；4.分子診斷(moleculardiagnosis)Restrictionfragmentlengthpolymorphism〔RFLP，限制性片段長(zhǎng)度多態(tài)性〕distinctionofDNAfrombacterialstrainsseparatedbypulsed-fieldgelelectrophoresis.Lanes1to3showSma1restrictionendonuclease-digestedDNAfrombacteriafromtwofamilymemberswithnecrotizingfasciitisandfromtheirphysician(pharyngitis).Lanes4to6arefromunrelatedStreptococcuspyogenesstrainsDNAprobe〔DNA探針〕analysisofvirus-infectedcells.SuchcellscanbelocalizedinhistologicallypreparedtissuesectionsusingDNAprobesconsistingofasfewasninenucleotidesorbacterialplasmidscontainingtheviralgenome.AtaggedDNAprobeisaddedtothesample.Theappropriatesubstrateisaddedtocolorthenucleiofvirallyinfectedcells.Insitulocalization〔原位雜交〕ofcytomegalovirus(CMV)infectionusingageneticprobe.CMVinfectionoftherenaltubulesofakidneyislocalizedwithabiotin-labeled,CMV-specificDNAprobeandisvisualizedbymeansofthehorseradishperoxidase-conjugatedavidinconversionofsubstrate,inamannersimilartoenzymeimmunoassay.多聚酶鏈反響DNAchip〔基因芯片〕16srRNA測(cè)序或全基因組測(cè)序蛋白組和質(zhì)譜檢測(cè)和定量抗原評(píng)價(jià)抗體反響評(píng)估是否暴露或免疫接種5.免疫學(xué)技術(shù)Analysisofantigensandantibodiesbyimmunoprecipitation〔免疫沉淀〕Immunofluorescenceandenzymeimmunoassaysforantigenlocalizationincells.Antigencanbedetectedbydirectassaywithantiviralantibodymodifiedcovalentlywithafluorescentorenzymeprobe,orbyindirectassayusingantiviralantibodyandchemicallymodifiedantiimmunoglobulin.Theenzymeconvertssubstratetoaprecipitate,chromophore,orlight.Immunofluorescencelocalizationofherpessimplexvirus–infectednervecellsinabrainsectionfromapatientwithherpesencephalitis.Flowcytometry.A,Theflowcytometerevaluatesindividualcellparametersasthecellsflowpastalaserbeamatratesofmorethan5000persecond.Cellsizeandgranularityaredeterminedbylightscattering(LS),andantigenexpressionisevaluatedbyimmunofluorescence(F),usingantibodieslabeledwithdifferentfluorescentprobes.GraphsBtoDdepictT-cellanalysisofanormalpatient.B,Light-scatteranalysiswasusedtodefinethelymphocytes(Ly),monocytes(Mo),andpolymorphonuclear(neutrophil)leukocytes(PMN).C,ThelymphocyteswereanalyzedforCD3expressiontoidentifyTcells(presentedinahistogram).D,CD4andCD8Tcellswereidentified.EachdotrepresentsoneTcellEnzymeimmunoassaysforquantitationofantibodyorantigen.A,Antibodydetection.1,Viralantigen,obtainedfrominfectedcells,virions,orgeneticengineering,isaffixedtoasurface.2,Patientserumisaddedandallowedtobindtotheantigen.Unboundantibodyiswashedaway.3,Enzyme-conjugatedantihumanantibody(E)isadded,andunboundantibodyiswashedaway.4,Substrateisaddedandconverted(5)intochromophore,precipitate,orlight.B,Antigencaptureanddetection.1,Antiviralantibodyisaffixedtoasurface.2,Aspecimenthatcontainsantigenisadded,andunboundantigeniswashedaway.3,Asecondantiviralantibodyisaddedtodetectthecapturedantigen.4,Enzyme-conjugatedantiantibodyisadded,washed,andfollowedbysubstrate(5),whichisconverted(6)intoachromophore,precipitate,orlight.Westernblotanalysis.Proteinsareseparatedbysodiumdodecylsulfate–polyacrylamidegelelectrophoresis(SDS),electroblottedontonitrocellulose(NC)paper,andincubatedwithantigenspecificorpatient’santisera(1°Ab)andthenenzyme-conjugatedantihumanserum(2°Ab).Enzymeconversionofsubstrateidentifiestheantigen.血清學(xué)診斷試驗(yàn)〔檢